Using Forced Degradation to Aid the Development of Biopharmaceutical Products

Abstract

Biotherapeutic products are lifesaving medicines for cancer, autoimmune and infectious diseases. In the last few years, the pharmaceutical industry has seen a massive spike in the development of biologics – over 2,700 biologics in development reported in 2018 alone. This trend fuels an ever-growing need for the development of new analytical methodologies to characterize the structure and function of biopharmaceutical products. The application of such new methodologies advances the understanding of their degradation mechanisms and provides useful knowledge in designing and meeting regulatory criteria. An overview of biopharmaceutical peptides, mAbs and biosimilars available on the market, along with their currently published analytical characterizations and typical instability mechanisms, are summarized in the first chapter. In the second chapter, we investigated the long-term stability of exenatide, a 39 amino acid GLP-1 receptor agonist peptide used to treat type 2 diabetes, under different experimental conditions. When exenatide was incubated at an elevated pH, rapid chemical and physical degradation occurred. Chemical degradation was characterized by a pH-dependent increase of deamidation impurities while physical degradation was mainly attributed to dimerization, aggregation and loss of α-helicity. The addition of excipients such as sucrose, mannitol and sorbitol showed a slight reduction of monomer loss at pH 7.5. In the third chapter, a comparability study between originator and biosimilar infliximab (Remicade® and Remsima™) was performed. Forced degradation was implemented to understand whether initial minor analytical differences could be amplified over the course of incubation. Some minor differences were found over incubation, including differences of heat capacity, intrinsic fluorescence, subvisible particulates, deamidation tendencies and fragmentation levels. Differences were not determined to be statistically significant and degradation mechanisms and kinetics were found to be highly similar. In the fourth chapter, a tandem mass-spectrometry method was employed to detect, identify and quantify disulfide bonds and related impurities (shuffled disulfide and trisulfide bonds) in originator and biosimilar pars of infliximab, rituximab and bevacizumab. Infliximab and bevacizumab biosimilars had higher levels of shuffled and trisulfide bonds relative to the originators, while rituximab biosimilar and originator had the similar levels of impurities. The bevacizumab and rituximab pairs were than incubated for 4 weeks at 37ºC to examine the kinetics of physical degradation by size exclusion chromatography and electrophoresis gels and disulfide shuffling by tandem mass-spectrometry. The two mAb pairs responded differently to forced degradation. The rituximab biosimilar had a slightly higher initial level of aggregation over incubation, relative to the originator, though degradation products were low and not exacerbated over the 4-week incubation. In contrast, the bevacizumab biosimilar had higher initial levels of protein aggregates and shuffled disulfide bonds, relative to the originator product, but also had exacerbated extent of aggregation and disulfide shuffling over the incubation than rituximab. This study indicates that originator and biosimilar pairs respond differently to forced degradation and that tandem mass-spectrometry is a useful tool to track the formation of covalent aggregates. Taken altogether, the thesis highlights the importance of the combination of classical analytical methodologies with new mass-spectrometry techniques to characterize instability mechanisms for peptide and mAb products subjected to forced-degradation conditions. The application of these techniques allows researchers, manufacturers and regulators to explore differences and similarities between reference biopharmaceutical products and their biosimilar (or generic) versions.